Treatment of Adult mdx Mice with Phosphorodiamidate Morpholino Oligomer Restores Cardiac Mitochondrial Energetics and Prevents the Dystrophic Cardiomyopathy

Monday, February 13, 2017 activation was shifted to more hyperpolarized potentials by ~20mV and doubled in effective valence. In non-facilitated a1C/b3 channels, VSD-II and -III are weaklycoupled to channel opening (Savalli et al., 2016 J Gen Physiol); pre-pulse-induced facilitation of their activation suggests that VDF engages these VSDs with the channel gate. This premise is supported by the finding that association of the a2d-1 subunit (which mimics facilitation; Platano et al., 2000 Biophys J) strongly couples VSD-II and -III to the channel gate (Pantazis et al., 2014 PNAS). Since VSD-I (i) is also potentiated by pre-pulses, but (ii) is weakly-coupled to channel opening in both non-facilitated a1C/b3 and ‘‘constitutively facilitated’’ a1C/b3/a2d channels, our work highlights it as a plausible candidate for the structural determinant of VDF. 1204-Pos Board B272 Treatment of Adult mdx Mice with Phosphorodiamidate Morpholino Oligomer Restores Cardiac Mitochondrial Energetics and Prevents the Dystrophic Cardiomyopathy Helena M. Viola1, Victoria P.A. Johnstone1, Abbie M. Adams2, Susan Fletcher2, Livia C. Hool1,3. 1 Physiology, The University of Western Australia, Crawley, Australia, 2 Centre for Comparative Genomics, Murdoch University, Murdoch, Australia, 3Victor Chang Cardiac Research Institute, Sydney, Australia. Duchenne muscular dystrophy (DMD) occurs due to the absence of dystrophin, and is associated with a fatal dilated cardiomyopathy. DMD-associated cardiomyopathy is underpinned by disrupted cytoskeletal architecture and poor energy metabolism. The L-type Ca2þ channel (LTCC) plays a critical role facilitating Ca2þ influx required for contraction, but also regulates mitochondrial function. This crosstalk is mediated via the cytoskeleton, and is disrupted in a mouse model of DMD (mdx). We previously demonstrated that treatment of pre-cardiomyopathic mdx mice with 10mg/kg/wk phosphorodiamidate morpholino oligomer (PMO) to induce skipping of exon 23 (M23D) for 24wks partially restored increases in mitochondrial membrane potential (Jm) and metabolic activity in response to activation of LTCC in myocytes isolated from pre-cardiomyopathic mice. Here we investigated the effect of a shortterm, but high PMO treatment dose, namely 120mg/kg/wk for 3wks, on alterations in Jm and metabolic activity. Treatment resulted in partial restoration of Jm and metabolic activity. Based on these findings we investigated whether a long-term and high PMO treatment dose would more effectively restore Jm and metabolic activity to wt levels. 24wk old pre-cardiomyopathic mdx mice were treated with 120mg/kg/wk for 19wks. Positive exon skipping was verified via RT-PCR, and immunohistochemistry confirmed the presence of dystrophin positive fibers within mdx heart tissue. PMO treatment completely restored Jm to wt levels (wt untreated: 30.954.2%, n=11; mdx untreated: 1.650.8%, n=8; mdx treated: 31.652.6%, n=7). PMO treatment also completely restored metabolic activity (assessed as formation of formazan from tetrazolium salt) (wt untreated: 48.956.4%, n=15; mdx untreated: 3.051.3%, n=11; mdx treated: 64.9514.0%, n=26). Furthermore, PMO treatment prevented development of the mdx cardiomyopathy assessed using serial echocardiography. These data indicate that treatment with M23D restores cardiac mitochondrial energetics in adult mdx mice. We propose that restoring mitochondrial function may play a vital role in mediating PMO-induced improvements in contractility. 1205-Pos Board B273 Stac Proteins Suppress Calcium Dependent Inactivation of Neuronal L-Type Ca2D Channels Alexander Polster, Stefano Perni, Kurt G. Beam. Physiology & Biophysics, University of Colorado, Denver, Aurora, CO, USA. Stac protein (named for its SH3 and cysteine rich domains) was first identified in brain 20 years ago, and is currently known to exist in three isoforms with specific tissue expression profiles. Transcripts for Stac1 and Stac2 are found in the cerebellum, forebrain, and eye, whereas transcripts for Stac3 are found at high levels in skeletal muscle and at low levels in the same three neuronally rich regions. However, the neuronal functions of Stac have been little investigated. Here, we tested the effects of Stac on currents via neuronal high-voltageactivated Ca2þ channels expressed together with their auxiliary subunits (b2a, a2-d1) in tsA201 cells. As described previously by others, we observed Ca2þ entry-dependent inactivation (CDI) for CaV1.2 and CaV1.3 (the predominant, neuronal L-type Ca2þ channels), and for the P/Q-type Ca2þ channel CaV2.1. CDI for CaV1.2 and CaV1.3 was suppressed almost completely by all three Stac isoforms, whereas CDI for CaV2.1 was not detectably altered. CDI is thought to depend on CaM constitutively bound to the CaV C-terminus, but based on co-expression of fluorescently tagged CaV1.2 and CaM, the Staccaused suppression of CDI did not involve the loss of bound CaM. Expression of CaV1.2 without auxiliary subunits resulted in small amplitude currents. These small amplitude currents displayed CDI which was similar to that when b2a and a2-d1 were also present, and which was suppressed by all three

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Stac isoforms. Thus, the Stac proteins appear to bind directly to CaV1.2 (and most likely to CaV1.3 as well) and to interfere with CDI without displacing CaM. Our results indicate that one likely function of neuronal Stac proteins is to tune Ca2þ entry via L-type channels. Supported by grants from NIH (AR052354 and AR070298) and MDA (176448) to KGB. 1206-Pos Board B274 Angiotensin II Stimulates L-Type Calcium Current in Mouse Atrial Myocytes by Affecting Caveolae-Housed Channels Marina Balycheva, Timothy J. Kamp, Alexey Glukhov. Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA. Angiotensin II (ANG II) plays an important role in atrial remodeling associated with atrial fibrillation and heart failure by affecting different processes including activation of abnormal Ca2þ influx. We hypothesized that caveolar localized L-type Ca channels (LTCCs) in atrial myocytes (AMs) are regulated by AngII. To examine thus further, we tested the effect of ANG II on L-type calcium current (ICa,L) in mouse (AMs). In wild type AMs, external application of 1 mM ANG II increased a peak of ICa,L by 122.9537.4% from baseline (n=7, P<0.01) and accelerated Ca2þ-dependent inactivation of ICa,L. Disruption of caveolae by 1-hour pre-treatment with 2 mM methyl-b-cyclodextrin (MbCD) decreased ICa,L density from 15.752.6 pA/pF, n=20 to 10.951.8 pA/pF, n=9, with no effect on activation and inactivation kinetics. Such ICa,L reduction may indicate the contribution of caveolae-housed channels. Importantly, MbCD prevented the stimulatory effect of ANG II on peak current and Ca2þ-dependent inactivation of ICa,L. Similarly, tamoxifen-induced conditional knock-out of caveolin-3 significantly reduced ICa,L density from 15.752.6 pA/pF, n=20 to 3.650.9 pA/pF, n=7 (P<0.005) and slowed Ca2þ-dependent inactivation. Importantly, AMs possess a highly variable membrane capacitance (73.458.1 pF, n=20) and ICa,L peak current (749.95177.9 pA, n=20) which correlated with the organization of transverse-axial tubular system (r2= 0.56) and possibly anatomical structure of the atria. Our findings indicate that ANG II enhances ICa,L primarily via localized stimulation of caveolae-housed LTCCs highlighting a potential significance of caveolae microdomains in ANG II dependent regulation of the atria. 1207-Pos Board B275 Stac3 Facilitated Expression of CaV1.1 in Xenopus Oocytes to Assess Functional Consequences of Hypopp Mutant CaV1.1-R528H Fenfen Wu, Steve C. Cannon. Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. Membrane expression of skeletal muscle L-type Ca2þ channels is notoriously poor in heterologous systems using non-muscle cells. Facilitated expression of CaV1.1 in tsA201 cells was recently achieved by co-expression of the Stac3 adapter protein (CH3 and cysteine-rich domain 3), and Stac3 may be directly involved in excitation-contraction coupling between CaV1.1 and RyR1 (Polster et al. PNAS 2015, 2016). We adopted this strategy to develop a CaV1.1 high-expression system in Xenopus oocytes. Because our goal is to define the functional consequences of disease-associated mutations in the voltage-sensor domain (VSD) of CaV1.1, we used the human cDNA for CaV1.1 (HsCD00347164). Oocytes were co-injected with cRNA for CaV1.1, a2d, and b1a subunits with or without Stac3. Currents recorded in 10 mM Ba2þ with the cut-open oocyte clamp were routinely 400 nA to 800 nA and occasionally exceeded 1 mA when Stac3 was co-expressed. The total gating charge movement was typically 500 pC. In the absence of Stac3, Ba2þ currents were less than 20 nA and in most cases were not detectable. CaV1.1 R528H is the most common mutation found in hypokalemic periodic paralysis (HypoPP). R528H channels expressed well in oocytes with peak Ba2þ currents about 20% lower than for WT and the voltage-dependence of activation was left-shifted by 8 mV. A small inward rectifying current, not conducted by the CaV1.1 pore, was detected for R528H channels; consistent with an anomalous gating pore current similar to that found in NaV1.4 HypoPP mutant channels. Supported by NIAMS of the NIH (AR063182).

Voltage-gated K Channels and Mechanisms of Voltage Sensing and Gating II 1208-Pos Board B276 Using Unnatural Amino Acids to Probe the Interaction between Tarantula Toxins and Voltage Sensing Domains in KV Channels Kanchan Gupta, Kenton J. Swartz. Molecular Physiology and Biophysics Section, NINDS, NIH, Bethesda, MD, USA. Peptide toxins folded into an inhibitor cystine knot (ICK) are found in the venoms of a variety of organisms (spider, scorpion, snakes, etc.), and many